The discovery and structure-activity relationships of pyrano[3,4-b]indole- based inhibitors of hepatitis C virus NS5B polymerase

Article abstract of DOI:10.1016/j.bmcl.2011.04.052

We describe the structure-activity relationship of the C7-position of pyrano[3,4-b]indole-based inhibitors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of the significantly more potent compounds 13 and 14

Full text of DOI:10.1016/j.bmcl.2011.04.052

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3227–3231
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The discovery and structure–activity relationships of pyrano[3,4-b]indole-based
inhibitors of hepatitis C virus NS5B polymerase
Randy W. Jackson a,d, , Matthew G. LaPorte b,d, , Torsten Herbertz e, Tandy L. Draper d, Janet A. Gaboury d,
⇑
⇑
Susan R. Rippin d, Ravi Patel d, Srinivas K. Chunduru f, Christopher A. Benetatos f, Dorothy C. Young f,
Christopher J. Burns d, Stephen M. Condon c,d,
⇑
a VenatoRx Pharmaceuticals Inc., 700 Pennsylvania Drive, Exton, PA 19341, USA
b University of Pittsburgh, Center for Chemical Methodologies and Library Development, Pittsburgh, PA 15260, USA
c TetraLogic Pharmaceuticals, 343 Phoenixville Pike, Malvern, PA 19355, USA
d Department of Medicinal Chemistry, ViroPharma Incorporated, 730 Stockton Drive, Exton, PA 19341, USA
e Department of Computational Chemistry, ViroPharma Incorporated, 730 Stockton Drive, Exton, PA 19341, USA
f Department of Biology, ViroPharma Incorporated, 730 Stockton Drive, Exton, PA 19341, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe the structure–activity relationship of the C7-position of pyrano[3,4-b]indole-based inhibi-
tors of HCV NS5B polymerase. Further exploration of the allosteric binding site led to the discovery of
the significantly more potent compounds 13 and 14.
Received 7 March 2011
Revised 11 April 2011
Accepted 12 April 2011
Available online 21 April 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
HCV NS5B polymerase
Pyranoindoles
Hepatitis C virus (HCV) infection continues to be a major global
health issue with recent total population infection estimates of
1-3%.1 HCV is the leading cause of advanced liver disease and
transplantation.2 Complications from chronic infection are respon-
sible for about 10,000–12,000 deaths/year in the U.S.2
Hepatitis C belongs to the Flaviviridae family of positive-single
stranded RNA viruses.6 The HCV genome encodes a 3000 amino
acid polyprotein which is processed into structural and non-struc-
tural proteins. One of the non-structural proteins that is essential
for viral replication is the NS5B RNA-dependent RNA polymer-
ase.7,8 The HCV NS5B is an attractive target owing to the success
of other antiviral agents that inhibit viral polymerases such as
the HIV non-nucleoside reverse transcriptase inhibitors (NNRTI’s).5
We have recently reported on a pyranoindole series of allosteric
HCV NS5B inhibitors that resulted in a potent analog demonstrat-
ing anti-viral activity in the chimeric mouse model of HCV infec-
tion.9 This communication will describe our optimization efforts
and structure-activity relationships at the C7-position of this novel
pyranoindole series of allosteric HCV NS5B enzyme inhibitors that
ultimately led to significantly more potent analogs.
Current therapies for HCV involve a combination of the nucleo-
side analog ribavirin and interferon-a 3
. This treatment regimen
causes unfavorable side effects which often results in poor patient
compliance. Furthermore, only about 40% of patients infected with
HCV achieve a sustained virologic response.4 Recently, pegylated
forms of interferon-
a (Pegasys™ and PEG-INTRON™) have im-
proved patient tolerance and response rates of over 50% have been
achieved. Sustained virologic responses have also been reported
with the combination of long-acting interferons with several
NS3/4a protease inhibitors of HCV.5 Some promising NS5B HCV
polymerase inhibitors are currently in various stages of develop-
ment. Similar to the successful anti-HIV ‘cocktail’ treatments,
future HCV therapies will most likely involve the combination of
agents that inhibit different viral targets. Clearly there is a medical
need for additional HCV antiviral agents.
Several thumb domain directed inhibitors of HCV NS5B have
been identified (Fig. 1).8 Compounds 1–4 occupy the same alloste-
ric binding site (Thumb Pocket II) of HCV NS5B with their acidic
functionalities participating in hydrogen bonding to the Ser476
and Tyr477 residues.
Another critical feature of these ligands is the ability to occupy a
hydrophobic region defined by residues Leu419, Trp528, Tyr477, and
Arg422
. Co-crystallographic analysis has determined that this
⇑
Corresponding author.
E-mail addresses: jackson@venatorx.com (R.W. Jackson), mgl12@pitt.edu (M.G.
hydrophobic ‘dimple’ on the NS5B protein is optimally occupied
by the C1 n-propyl group of 1.9 Further examination of the protein
LaPorte), scondon@tetralogicpharma.com (S.M. Condon).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.04.052

3228
R. W. Jackson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3227–3231
Our strategy relied on a flexible synthetic route that allows for
introduction of a variety of substitutions with the initial focus on
carboxamides as shown in Scheme 1.
Representative assay results for the carboxamide series are
shown in Table 1. All of these analogs were prepared as racemates.
These amide analogs are generally less active against the HCV
NS5B enzymes than the unsubstituted derivative (1), although 9e
and 9g appear to be equipotent to 1.10 Disubstituted amides such
as 9b are not well tolerated.
S
CN
Me
CO2H
O
N
O
CO2H
OH
N
H
1 (HCV-371)
2
We were encouraged by these results because they demon-
strated that the C7-substituted pyranoindoles were tolerated in
the thumb domain binding site. However, the C7-containing benz-
amides had poor activity in the cell-based HCV replicon assay.
We reasoned that the decrease in cellular activity was related to
the higher polar surface areas (PSA) of the amides relative to 1,11
and that replacement of the amide linker with a less polar func-
tionality may restore the cellular activity. Therefore, we turned
our attention to preparing a series of C7-substituted ethers
(Scheme 2). The benzoic acid functionality in 8 was converted into
the aldehyde derivative (10) which then underwent a Baeyer–Vil-
liger oxidation to provide phenol 11. The racemic ethers (12) were
formed by either standard alkylation or Mitsunobu reactions.
Representative C7-ether analogs were significantly more potent
against the HCV polymerase enzymes compared to 1 (Table 2).
Phenol 12a was approximately twice as potent as 1 against BK
and BB7 enzymes but possessed no appreciable cellular activity.
The C7 methyl ether (12b) was similarly active against the
polymerase and equipotent to 1 in the cell-based replicon assay.
Optimal tether lengths were observed through preparation of the
ethyl, n-propyl and the methoxyethyl analogs (12c, 12d, and 12f,
respectively). These analogs all demonstrated similar cellular
activities as well as improved affinities against the isolated NS5B
enzymes suggesting that these compounds were accessing the
hydrophobic shelf. Alkyl branching (12e) or incorporation of a
basic amine (12g) were not well tolerated.
Et
Me
OH
O
N
N
N
CO2H
O
N
N
N
F3C
O
Me
Et
Cl
3
4
Cl
Figure 1. Reported allosteric thumb pocket II HCV NS5B inhibitors.
Further SAR development of the C7 ethers showed that hetero-
aromatic substrates tethered via an alkyl chain were consistently
among the most potent compounds (Table 3). The 2-pyridylmethyl
compound (12i) was significantly more active than 1 in the repli-
con assay. The 4-pyridylmethyl analog was slightly less active,
and the addition of an extra methylene unit into the tether resulted
in a 10-fold drop in enzyme inhibition. Alkyl-substitution on the
pyridine ring was generally well- tolerated but did not provide sig-
nificant advantages (not shown), nor did addition of a second
nitrogen (12k). Five-membered heteroaromatic substituents were
also highly potent inhibitors of the HCV polymerase. The 5-methy-
lisoxazole (12l) was among the most potent with sub-micromolar
activity in the cell-based replicon assay. Co-crystallographic analy-
Figure 2. Crystal structure of NS5B with tetrahydro[3,4-b]indole inhibitor. Elec-
trostatic potential representation: Red-negative charge; Blue-positive charge;
White-non-polar; Ser476 & Tyr477 highlighted in Green.
reveals a hydrophobic shelf near the aromatic core of the pyrano-
indole (Fig. 2). We hypothesized that this shelf may provide an
opportunity for additional protein-ligand interactions affording
compounds with enhanced binding affinity. Structural analysis
suggested that substitution at the C7 position of the pyranoindole
nucleus would provide the required trajectory for accessing this
hydrophobic region (See Fig 2).
Br
Br
a-d
O
CO2Et
e-f
HO2C
NO2
BnO2C
NHNH2
N
H
BnO2C
Me
Me
Me
7
5
6
CN
Me
CN
Me
O
O
CO2H
CO2Et
R2
g-h
i-j
N
R1
N
N
H
HO2C
H
O
8
9
Scheme 1. Reagents: (a) N,N-dibromodimethyl-hydantoin, H2SO4, 99%; (b) BnBr, DIPEA, THF, 84%; (c) Fe, NH4Cl, H2O, 100 °C, 92%; (d) NaNO2, HCl, then SnCl2, 93%; (e)
Dihydrofuran, ZnCl2, H2O, ethylene glycol, 100 °C, 27%; (f) Ethyl-3-oxapentanoate, BF3ÁOEt2, DCM, 84%; (g) CuCN, NMP, 190 °C, 82%; (h) H2, Pd(OH)2, MeOH/EtOAc, 96%; (i)
R2R1NH, HATU, NMM, DCM; (j) NaOH, H2O, EtOH.

R. W. Jackson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3227–3231
3229
Table 1
C7-Pyranoindole carboxamides
CN
O
CO2H
R2
N
R1
N
H
O
Me
Compd
R1
R2
HCV pol (BK) IC50
,
lM
HCV pol (BB7) IC50
,
lM
HCV NS5A EC50
,
lM
PSA
86.1
115.2
106.4
115.2
115.2
1
—
H
Me
H
H
—
0.3
1.3
16
3
0.98
0.4
0.97
20
2
0.67
13.1
9a
9b
9c
9d
Me
Me
nPr
Bn
>100
>100
>100
>100
O
9e
H
0.54
0.3
56
133.6
O
N
9f
H
H
1.2
0.64
0.56
>100
>100
128.1
143.9
N
9g
0.86
NH
⁄A description of the assay conditions used to determine IC50’s and EC50’s can be found in Ref. 9d.
CN
CN
CN
Me
O
CO2Et
O
O
CO2Et
c-d
CO2H
c-d
e-f
8
N
H
OHC
N
H
N
H
HO
RO
Me
Me
10
12
11
Scheme 2. Reagents and conditions: (a) BH3ÁTHF, THF, 77%; (b) MnO2, C6H6, 1,2-DCE, reflux, 96%; (c) SeO2 (0.1 equiv), H2O2, t-BuOH, H2O, TFA; (d) K2CO3, MeOH, 73%-2 steps;
(e) R-X, K2CO3, DMF or ROH, DIAD, PPh3, DCM; (f) NaOH, H2O, EtOH.
Table 2
Initial C7-alkoxy-pyranoindoles
CN
O
CO2H
N
H
RO
Me
Compound
R
HCV pol (BK) IC50
,
l
M
HCV pol (BB7) IC50
,
lM
HCV NS5A EC50
,
lM
PSA
86.1
106.3
95.3
95.3
95.3
95.3
1
—
H
CH3
Et
nPr
iPr
0.3
0.4
0.2
0.13
0.07
0.07
0.22
13.1
47
20
3.5
2.9
19.7
12a
12b
12c
12d
12e
0.26
0.36
0.11
0.07
0.48
MeO
12f
0.04
0.09
4.9
104.6
12g
1.1
0.95
26
98.6
N
sis has determined that the pendant isoxazole group of 12l occu-
pies the proximal hydrophobic shelf (See Fig 3). Dimethylpyrazole
12m had an EC50 of 0.76 lM in the replicon assay. The 2-pyrazol-
ylethyl analog (12n) was among the most active analogs prepared
in this series. The incorporation of a (S)-methyl group adjacent to
the pyrazole ring of 12n increased potency even further, generat-
(S)-sec-butyl afforded susbstrates with improved potencies.9b To-
wards this end we combined the C7-ethyl-linked pyrazole group
with the (S)-sec-butyl substitution (13 and 14, Table 4) that pro-
duced significant increases in cellular activities.9a The pyrazole
containing analogs are the most potent pyranoindole-based HCV
inhibitors prepared to date, and 13 has been shown to be effica-
cious in a chimeric mouse model of hepatitis C infection.9a
We have described the evolution and SAR of a C7-substituted
pyranoindole-based series of HCV NS5B polymerase inhibitors.
Through structure-based design, we have identified and detailed
ing a compound (12o) with a replicon EC50 of 0.12
stereomer 12p was less active (EC50 = 0.34 M).
lM while dia-
l
Parallel efforts of optimizing the C1 position of the pyranoin-
dole demonstrated that replacement of the n-propyl group with

3230
R. W. Jackson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3227–3231
Table 3
C7-Heteroaryl-containing pyranoindoles
CN
O
CO2H
N
H
RO
Me
Compound
R
BK IC50
0.3
(lM)
BB7 IC50
0.4
(lM)
HCV NS5A EC50
13.1
(l
M)
PSA
86.1
1
—
N
12i
12j
0.02
0.31
0.02
0.23
0.39
8.3
108.2
108.2
N
N
N
12k
0.019
0.006
0.02
1.6
121.1
121.4
(1R)-12l
0.008
0.48
N
N
Me
Me
O
N
(1R)-12m
0.03
0.02
0.76
113.2
Me
N
N
(1R)-12n
(1R)-12o
0.005
0.003
0.005
0.003
0.88
0.12
113.2
113.2
N
N
Me
N
N
(1R)-12p
0.007
0.007
0.34
113.2
Me
Table 4
C1-sec-Butyl analogs
Compound
Structure
BK IC50
0.012
(l
M)
BB7 IC50
0.003
(lM)
HCV RNA EC50 (lM)
CN
O
O
CO2H
CO2H
N
N
13
14
0.02
N
H
O
O
Me
CN
N
N
0.002
0.002
0.02
N
H
Me
Me
the preparation and biological activities of these potent inhibitors.
Significant improvements in enzyme and cellular potencies were
realized by incorporating ether substitutions at the C7-position
of the pyranoindole nucleus. Ultimately, we were able to identify
ethoxy- pyrazole analogs 13 and 14 that were significantly more
potent than 1.
Acknowledgements
We wish to thank our collaborators at Wyeth for their
assistance.
References and notes
1. (a) Shepard, C. W.; Finelli, L.; Alter, M. J. Lancet Infect. Dis. 2005, 5, 558; (b) Alter,
M. J.; Moran, D. K.; Nainan, O. V.; McQuillan, G. M.; Gao, F.; Moyer, L. A.; Kaslow,
R. A.; Margolis, H. S. N. Eng. J. Med. 1999, 341, 556.
2. (a) Hanazaki, K. Curr. Med. Chem.; Anti-Infect. Agents 2003, 2, 103; (b) Lauer, G.
M.; Walker, B. D. N. Eng. J. Med. 2001, 345, 41; (c) Cohen, J. Science 1999, 285,
Figure 3. Crystal structure of NS5B with tetrahydro[3,4-b]indole inhibitor 12l.
Electrostatic potential representation: Red-negative charge; Blue-positive charge;
White-non-polar.

R. W. Jackson et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3227–3231
3231
26; (d) Idéo, G.; Bellobuono, A. Curr. Pharm. Design 2002, 8, 959; (e) Di Bisceglie,
A. M.; McHutchison, J.; Rice, C. M. Hepatology 2002, 35, 224; (f) Samuel, C. E.
Clin. Microbiol. Rev. 2001, 14, 778.
J.; Linton, A.; Gonzalez, J.; Jewell, T.; Patel, L.; Ludlum, S.; Drowns, M.;
Rahavendran, S. V.; Skor, H.; Hunter, R.; Shi, S. T.; Herlihy, K. J.; Parge, H.;
Hickey, M.; Yu, X.; Chau, F.; Nonomiya, J.; Lewis, C. J. Med. Chem. 2009, 52, 1255;
(e) Beaulieu, P. L. Curr. Opin. Invest. Drugs 2007, 8, 614.
3. (a) Hoofnagle, J. H. N. Eng. J. Med. 2009, 360, 1899; (b) Youngster, S.; Wang, Y.-
S.; Grace, M.; Bausch, J.; Bordens, R.; Wyss, D. F. Curr. Pharm. Design 2002, 8,
2139; (c) Tan, S.-L.; Pause, A.; Shi, Y.; Sonenberg, N. Nat. Rev. Drug Discovery
2002, 1, 867.
4. For a recent medicinal chemistry perspective on HCV, See: Gordon, C. P.; Keller,
P. A. J. Med. Chem. 2005, 48, 1.
5. For HCV NS5B inhibitors demonstrating antiviral effects in early clinical trials,
see: (a) Flisiak, R.; Parfieniuk, A. Expert Opin. Invest. Drugs 2010, 19,
63; (b) Kwong, A. D.; McNair, L.; Jacobson, I.; George, S. Curr. Opin. Pharmacol.
2008, 8, 522; (c) Asselah, T.; Benhamou, Y.; Marcellin, P. Liver Inter. 2009, 29,
57.
6. (a) Bartenschlager, R.; Lohmann, V. J. Gen. Virology 2000, 81, 1631; (b)
Moradpour, D.; Brass, V.; Gosert, R.; Wölk, B.; Blum, H. E. Trends Mol. Med.
2002, 8, 476.
9. (a) LaPorte, M. G.; Jackson, R. W.; Draper, T. L.; Gaboury, J. A.; Galie, K.;
Herbertz, T.; Hussey, A. R.; Rippin, S. R.; Benetatos, C. A.; Chunduru, S. K.;
Christensen, J. S.; Coburn, G. A.; Rizzo, C. J.; Rhodes, G.; O’Connell, J.; Howe, A. Y.
M.; Mansour, T. S.; Collett, M. S.; Pevear, D. C.; Young, D. C.; Gao, T.; Tyrrell, D. L.
J.; Kneteman, N. M.; Burns, C. J.; Condon, S. M. ChemMedChem 2008, 3, 1508; (b)
LaPorte, M. G.; Draper, T. L.; Miller, L. E.; Blackledge, C. W.; Leister, L. K.;
Amparo, E.; Hussey, A. R.; Young, D. C.; Chunduru, S. K.; Benetatos, C. A.;
Rhodes, G.; Gopalsamy, A.; Burns, C. J.; Condon, S. M. Bioorg. Med. Chem. Lett.
2010, 20, 2968; (c) Gopalsamy, A.; Lim, K.; Ciszewski, G.; Park, K.; Ellingboe, J.
W.; Bloom, J.; Insaf, S.; Mansour, T. S.; Krishnamurthy, G.; Damarla, M.; Pyatski,
Y.; Ho, D.; Howe, A. Y. M.; Orlowski, M.; Feld, B.; O’Connell, J. J. Med. Chem.
2004, 47, 6603; (d) Howe, A. Y. M.; Bloom, J.; Baldick, C. J.; Benetatos, C. A.;
Cheng, H.; Christensen, J. S.; Chunduru, S. K.; Coburn, G. A.; Feld, B.; Gopalsamy,
A.; Gorczyca, W. P.; Herrmann, S.; Johann, S.; Jiang, X.; Kimberland, M. L.;
Krisnamurthy, G.; Olson, M.; Orlowski, M.; Swanberg, S.; Thompson, I.; Thorn,
M.; Del Vecchio, A.; Young, D. C.; van Zeijl, M.; Ellingboe, J. W.; Upeslacis, J.;
Collett, M.; Mansour, T. S.; O’Connell, J. F. Antimicrob. Agents Chemother. 2004,
48, 4813.
7. (a) Wu, J. Z.; Hong, Z. Curr. Drug Targets 2003, 3, 207; (b) Walker, M. P.; Hong, Z.
Curr. Opin. Pharmacol. 2002, 22, 1; (c) Zhang, X. Idrugs 2002, 5, 154; (d)
Bartenschlager, R. Antiviral Chem. Chemother. 1997, 8, 281; (e) Dymock, B. W.;
Jones, P. S.; Wilson, F. X. Antiviral Chem. Chemother. 2000, 11, 79; (f) De Clercq,
E. Nat. Rev. Drug Discovery 2007, 6, 1001.
8. For recent reviews on HCV RNA RdRp inhibitors, see: (a) Lévêque, V. J.-P.;
Wang, Q. M. Cell. Mol. Life Sci. 2002, 59, 909; (b) De Francesco, R.; Tomei, L.;
Altamura, S.; Summa, V.; Migliaccio, G. Antiviral Res. 2003, 58, 1; (c) Condon, S.
M.; LaPorte, M. G.; Herbertz, T. Curr. Med. Chem. 2005, 4, 99; (d) Li, H.; Tatlock,
10. The IC50’s are an average of multiple experiments with the standard deviations
generally within 10% of this value.
11. Physicochemical properties calculated using Schrödinger 2010 Suite, Maestro
9.1-QikProp 3.3; Schrödinger, LLC: New York.